1. Field of the Invention:
The present invention relates to a novel method for hydrolysis of esters and in particular to methods for making polypeptides via ester hydrolysis. The present invention uses novel enzymes to catalyze the ester hydrolysis which novel enzymes are selected and modified to have a amide hydrolysis rate which is significantly less than the ester hydrolysis rate. The invention further relates to enzymes and peptides made by the method of the invention.
2. Background Art
Many enzymes are known to catalyze ester hydrolysis. This type of hydrolysis reaction is useful, for example, in combining an ester of a given amino acid or peptide with another amino acid or peptide to form a polypeptide. It is hypothesized that a binding site in such enzymes exists which catalyzes this reaction. Likewise, these enzymes also catalyze amide hydrolysis, the reverse reaction, which acts as the yield limiting equilibrium reaction. Proteases, notably the serine proteases and thiol proteases are known to catalyze reactions which involve the catalytic triad of the enzyme. (Enzyme Structure and Mechanism, 2nd Edition, 1985, pp. 405-413). It is further understood that the intermediates in the reaction are probably stabilized by the "oxyanion hole" (Proteases and Biological Control, Reichet et al, 1975, pp. 5-27). The total mechanism of ester/amide hydrolysis is fairly well understood. It is known that interfering with this mechanism, for example by methylating a His 57 in the catalytic triad of chymotrypsin, that the reaction rates drop by a factor of 5,000-200,000. It has been assumed that this dropping activity is essentially equal for rates of hydrolysis for both amides and esters.
In Phil. Trans. R. Soc. Lond. A 317, 415-423 (1986), changes were also made for the first time in the amino acid residue comprising the oxyanion hole. In this instance, Ans-155 in subtilisin was modified to Thr, His, Gln and Asp by using a site specific mutagenisis of cloned subtilisin from B. Amyloliquefaciens. A large decrease in substrate turnover (K.sub.cat) of 200-4,000 fold was observed for an amide substrate. However this was the only sustrate tested. Ester hydrolysis was not measured. Based on the mechanism and the prior art it would be logical to assume that the ester hydrolysis substrate turnover rate would also be reduced and the reduction would be proportional to the decrease amide hydrolysis. When selecting an enzyme for use in ester hydrolysis it would be useful to block or at least substantially reduce the equilibrium reaction toward amide hydrolysis. One way of accomplishing this is to have some method of removing the product before the amide hydrolysis can occur. Another theoretical way of accomplishing this would be to change the K.sub.cat /K.sub.cat ratio for ester substrate verses amide substrate such that amide K.sub.cat is reduced to a greater extent than ester K.sub.cat. Prior to applicant's invention the only possible way to alter one K.sub.cat more or less than the other K.sub.cat was to alter the reaction conditions i.e. pH. For example, in Carlsberg Res. Common Vol. 45, p. 237-247, 1980, Carboxypeptidase Y was used to catalyze peptide synthesis. The equilibrium of the competing reactions was changed by performing the reaction at high pH (i.e.&gt;9). These conditions are obviously not advantageous for most enzymes which operate at lower pH, normally around neutral. It is interesting to note even further that the author notes that a single desired product is still not produced in high yield.
Accordingly, a method which uses an enzyme which is selective for ester hydrolysis over amide hydrolysis and operates at a variety of pH and other conditions would be useful.